JP5795259B2 - Primers and probes for detecting human papillomavirus and human beta globin sequences in test samples - Google Patents

Description

  The present invention relates to primers, probes, primer sets, primers and probe sets, methods and kits for detecting human papillomavirus, human beta globin sequences and human papillomavirus and human beta globin sequences in a test sample.

  Papillomaviruses are DNA viruses that infect human and animal skin and mucous membranes. About 130 types of human papillomavirus (HPV) have been identified, of which 30-40 are transmitted by sexual contact and infect the anogenital region. Some of these HPV types cause genital warts and some do not cause any significant signs of infection. At least 14 HPV types, ie types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68, have been associated with a high risk for cervical cancer. The detection of these high-risk HPVs is important in preventing cervical cancer.

  All HPV-type genomes are similarly organized. Several early (E) and late (L) proteins are specifically encoded. DNA replication requires E1 and E2 proteins. Replication of the viral genome in the upper layer of the epithelium requires E4 and E5 proteins. E6 and E7 proteins are oncogenic and act cooperatively to immortalize cells and induce genomic instability. L1 and L2 proteins form a viral capsid and are expressed late in infection in the upper layer of the epithelium. Another part of the genome, the long-control-region (LCR), contains most of the regulatory DNA sequences necessary for proper replication of the viral genome and expression of the viral gene.

  Various methods have been devised for detecting high risk HPV. Many are based on the detection of unique sequences within the HPV genome. For example, DNA or RNA probes complementary to a portion of the HPV type 35 gene are described as useful in screening for the presence of this HPV type in a test sample, for example, in US Pat. No. 4,849,332. ing. Other probe sequences useful for detecting oncogenic HPV types are disclosed in US Pat. No. 6,265,154. US Pat. No. 5,705,627 describes the use of polymerase chain reaction (PCR) to amplify and detect HPV DNA using degenerate or mixed consensus primers, followed by genotype-specific DNA probes. Teaching type discrimination using mixtures. Other examples using consensus primers are US Pat. No. 5,364,758 and Kleter, B. et al. Et al., Am. J. et al. of Pathology, 1998, 153 (6): 1731-39. In addition, many of the methods known in the art also involve detecting human beta globin sequences in a test sample.

US Pat. No. 4,849,332 US Pat. No. 6,265,154 US Pat. No. 5,705,627 US Pat. No. 5,364,758

Kleter, B.M. Et al., Am. J. et al. of Pathology, 1998, 153 (6): 1731-39

  As mentioned above, various methods for detecting high risk HPV types are known in the art. Despite the existence of such a method, (1) multiple HPV genotypes can be detected in a single reaction, while at the same time the detection of one specific genotype can be distinguished from the detection of other genotypes, There is a need in the art for new methods that provide (2) no cross-reactivity between HPV types, (3) provide robust clinical sensitivity and specificity, and (4) provide high throughput and efficient processing. .

In one embodiment, the present invention relates to human papillomavirus (HPV) types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 in a test sample. It relates to a primer for amplification. The primer has a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and their complements.

  In another embodiment, the present invention relates to a probe for detecting HPV 16, 18, 31, 35, 39, 45, 51, 52, 58, 59 or 66 in a test sample. The probes are SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and their Having a sequence selected from the group consisting of complements.

  In yet another embodiment, the present invention relates to primers for amplifying human beta globin sequences in a test sample. The primer has the sequence of SEQ ID NO: 6, SEQ ID NO: 7, the complement of SEQ ID NO: 6, the complement of SEQ ID NO: 7, or any combination thereof.

  In yet another embodiment, the present invention relates to a probe for detecting human beta globin sequences in a test sample. The probe has the sequence of SEQ ID NO: 22 or its complement.

In yet another embodiment, the present invention amplifies HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 in the test sample. It relates to the primer set. The primer set includes:
(A) at least one forward primer having a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, their complements and any combination thereof; and (b) SEQ ID NO: 4, At least one reverse primer having a sequence selected from the group consisting of SEQ ID NO: 5, their complements, and any combination thereof.

  In particular, the primer set can include forward primers having sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 or their complements. The primer set can include reverse primers having the sequences of SEQ ID NO: 4 and SEQ ID NO: 5 or their complements. Alternatively, the primer set includes SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 or their forward primers, and SEQ ID NO: 4 and SEQ ID NO: 5 or their reverse primers. Can be included.

In yet another embodiment, the present invention detects HPV types 16, 18, 31, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 in the test sample. The primer and probe set. The primer and probe set are:
(A) three forward primers having the sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 or their complements, and two reverses having the sequences of SEQ ID NO: 4 and SEQ ID NO: 5 or their complements A primer,
(B) SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 , And 14 probes having the sequences of SEQ ID NO: 20 and SEQ ID NO: 21 or their complements.

In yet another embodiment, the invention relates to a primer and probe set for detecting human beta globin in a test sample. The primer and probe set are:
(A) a forward primer having the sequence of SEQ ID NO: 6 or its complement, and a reverse primer having the sequence of SEQ ID NO: 7 or its complement;
(B) a probe having the sequence of SEQ ID NO: 22 or its complement.

In yet another embodiment, the present invention provides one or more of HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 in the test sample. It relates to a method for detecting. The method
(A) Three forward primers having the sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 or their complements, and two reverse primers having the sequences of SEQ ID NO: 4 and SEQ ID NO: 5 or their complements And a test sample under amplification conditions to produce a first target sequence,
(B) a first target sequence and an indication of the presence of one or more of HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 in the test sample Detecting hybridization between at least one probe as, wherein the probe comprises SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, It has the sequence of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21 or their complements.

  In the method, the amplification conditions include subjecting the test sample to an amplification reaction performed in the presence of a suitable amplification reagent. The amplification reaction can also include using PCR, real-time PCR (such as, but not limited to, Taq-Man® assay) or reverse transcription PCR (RT-PCR).

  In said method, at least one probe is labeled with a detectable label. As is known in the art, the detectable label can be bound directly to at least one probe. Alternatively, the detectable label can be indirectly bound to at least one probe. Furthermore, the detectable label may be directly detectable. Alternatively, the detectable label can be indirectly detectable. For example, the detectable label can include a fluorescent moiety attached to the 5 'end of at least one probe. Furthermore, the at least one probe can further comprise a quenching moiety attached to its 3 'end.

The method further includes:
(A) contacting a test sample with a forward primer having the sequence of SEQ ID NO: 6 or its complement and a reverse primer having the sequence of SEQ ID NO: 7 or its complement under amplification conditions to generate a second target sequence;
(B) detecting hybridization between the second target sequence and a probe having the sequence of SEQ ID NO: 22 or its complement as an indicator of the presence of human beta globin in the test sample.

In yet another embodiment, the present invention relates to a method for detecting and / or identifying HPV type 16, type 18 or both HPV types 16 and 18 in a test sample. The method
(A) Three forward primers having the sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 or their complements, and two reverse primers having the sequences of SEQ ID NO: 4 and SEQ ID NO: 5 or their complements And a test sample under amplification conditions to produce a first target sequence,
(B) The first target sequence and the following:
(I) a first probe having the sequence of SEQ ID NO: 8 or its complement as an indicator of the presence of HPV type 16 (the first probe is labeled with a first detectable label),
(Ii) a second probe having the sequence of SEQ ID NO: 9 or its complement as an indicator of the presence of HPV type 18 (the second probe is labeled with a second detectable label, and the second detectable label is , A detectable label different from the first detectable label).
(Iii) HPV 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 or 68 as an indicator of the presence of the type SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, One or more additional probes having sequences of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21 or their complements (such as Each of the one or more additional probes is labeled with the same third detectable label, and the third detectable label is a different detectable label than the first detectable label and the second detectable label. .)).

  In the method, the amplification conditions include subjecting the test sample to an amplification reaction performed in the presence of a suitable amplification reagent. The amplification reaction can also include using PCR, real-time PCR (such as, but not limited to, Taq-Man® assay) or RT-PCR.

In yet another embodiment, the invention relates to a method for detecting human beta globin in a test sample. The method
(A) contacting a test sample with a forward primer having the sequence of SEQ ID NO: 6 or its complement and a reverse primer having the sequence of SEQ ID NO: 7 or its complement under amplification conditions to generate a first target sequence;
(B) detecting hybridization between the first target sequence and a probe having the sequence of SEQ ID NO: 22 or its complement as an indicator of the presence of human beta globin in the test sample.

  In the method, the amplification conditions include subjecting the test sample to an amplification reaction performed in the presence of a suitable amplification reagent. The amplification reaction can also include using PCR, real-time PCR (such as, but not limited to, Taq-Man® assay) or RT-PCR.

  In said method, at least one probe is labeled with a detectable label. The detectable label can be bound directly to at least one probe. Alternatively, the detectable label can be indirectly bound to at least one probe. Furthermore, the detectable label may be directly detectable. Alternatively, the detectable label can be indirectly detectable. For example, the detectable label can include a fluorescent moiety attached to the 5 'end of at least one probe. Furthermore, the at least one probe can further comprise a quenching moiety attached to its 3 'end.

The method further includes:
(A) Three forward primers having the sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 or their complements, and two reverse primers having the sequences of SEQ ID NO: 4 and SEQ ID NO: 5 or their complements And a test sample under amplification conditions to generate a second target sequence,
(B) a second target sequence and an indication of the presence of one or more of HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 in the test sample SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, Detecting hybridization between at least one probe having the sequence of SEQ ID NO: 20 and SEQ ID NO: 21 or their complements.

In yet another aspect, the present invention is for detecting HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 in a test sample. About the kit. The kit
(A) at least one forward primer having a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, their complements and any combination thereof;
(B) comprising at least one reverse primer having a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, their complements and any combination thereof, and (c) an amplification reagent.

  The kit may further comprise at least one probe, wherein the at least one probe is SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 , SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21 or their complements.

  The kit further includes SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 18. Probes having the sequence of SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21 or their complements

In yet another aspect, the present invention is for detecting HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 in a test sample. It relates to a primer and a probe kit. The kit
(A) Three forward primers having the sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 or their complements, and two reverse primers having the sequences of SEQ ID NO: 4 and SEQ ID NO: 5 or their complements ,
(B) SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 , 14 probes having the sequence of SEQ ID NO: 20 and SEQ ID NO: 21 or their complements, and (c) an amplification reagent.

  The kit further comprises a forward primer having a sequence of SEQ ID NO: 6 or its complement and a reverse primer having a sequence of SEQ ID NO: 7 or its complement for detecting human beta globin in a test sample; Or a probe having a sequence of its complement.

In yet another aspect, the invention relates to a primer and probe kit for detecting human beta globin in a test sample. The kit
(A) a forward primer having the sequence of SEQ ID NO: 6 or its complement and a reverse primer having the sequence of SEQ ID NO: 7 or its complement;
(B) a probe having the sequence of SEQ ID NO: 22 or its complement, and (c) an amplification reagent.

The kit further includes
(D) three forward primers having the sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 or their complements, and two reverse primers having the sequences of SEQ ID NO: 4 and SEQ ID NO: 5 or their complements And (e) SEQ ID NO: 8, SEQ ID NO: 9 for detecting HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 in the test sample , SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21 or those 14 probes having the sequence of their complements can be included.

FIG. 1 relates to the relationship between the amount of spiked cells from a cultured HPV positive cell line and the beta globin signal as described in Example 4 and a population of 1206 patient cervical samples. The distribution of the number of beta globin cycles is shown. FIG. 2 shows the distribution of the number of beta globin cycles for a population of 1206 patient cervical samples, as described in Example 4. Quartile points for the relationship described in FIG. FIG. 3A shows a comparison of analytical performance of a primer mixture comprising SEQ ID NOs: 1-5 and GP5 + and GP6 + primers (SEQ ID NOs: 23-24) as described in Example 5. FIG. 3B shows a comparison of the analytical performance of the primer mixture comprising SEQ ID NOs: 1-5 and GP5 + and GP6 + primers (SEQ ID NOs: 23-24) as described in Example 5. FIG. 3C shows a comparison of the analytical performance of the primer mixture comprising SEQ ID NOs: 1-5 and GP5 + and GP6 + primers (SEQ ID NOs: 23-24) as described in Example 5. FIG. D shows a comparison of the analytical performance of a primer mixture comprising SEQ ID NOs: 1-5 and GP5 + and GP6 + primers (SEQ ID NOs: 23-24) as described in Example 5. FIG. 4A shows probes of the present invention (SEQ ID NOs: 8-10, 16 and 19) specific for HPV types 16, 18, 31, 52 and 59 and US Pat. No. 6,6, as described in Example 5. A comparison of analytical performance with probe sequences for the same HPV type disclosed in 265,154B1 is shown. FIG. 4B shows the probe of the present invention (SEQ ID NOs: 8-10, 16 and 19) specific for HPV types 16, 18, 31, 52 and 59 and US Pat. No. 6,6, as described in Example 5. A comparison of analytical performance with probe sequences for the same HPV type disclosed in 265,154B1 is shown. FIG. 4C shows a probe of the invention (SEQ ID NOs: 8-10, 16 and 19) specific for HPV types 16, 18, 31, 52 and 59 and US Pat. No. 6,6, as described in Example 5. A comparison of analytical performance with probe sequences for the same HPV type disclosed in 265,154B1 is shown. FIG. 4D shows the probe of the present invention (SEQ ID NOs: 8-10, 16 and 19) specific for HPV types 16, 18, 31, 52 and 59 and US Pat. No. 6,6, as described in Example 5. A comparison of analytical performance with probe sequences for the same HPV type disclosed in 265,154B1 is shown. FIG. 4E shows the probe of the present invention (SEQ ID NOs: 8-10, 16 and 19) specific for HPV types 16, 18, 31, 52 and 59, as described in Example 5, and US Pat. A comparison of analytical performance with probe sequences for the same HPV type disclosed in 265,154B1 is shown.

DETAILED DESCRIPTION The present invention is used to amplify and / or detect HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 in a test sample. Relates to primers, probes, primer sets and primers and probe sets that can be made. The present invention also provides HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 in test samples using the primer and probe sets herein. It relates to the detection method. Furthermore, the present invention also relates to primers, probes and primers and probe sets that can be used to amplify and / or detect human beta globin sequences in a test sample. Primers and probes used to amplify and / or detect human beta globin in a test sample can be used to generate an internal control amplicon in the HPV assay. Furthermore, the present invention also relates to a method for detecting human beta globin sequences in a test sample using the primers and probe sets herein. The present invention is also for detecting HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 and / or human beta globin sequences in a test sample. About the kit.

  The primer and probe sets of the present invention provide robust clinical sensitivity and specificity. Also, the primer and probe sets herein show no cross-reactivity between HPV types. Furthermore, the primer and probe set of the present invention can detect multiple HPV genotypes in a single reaction, while simultaneously distinguishing the detection of one genotype from the detection of another genotype (eg, partial genotyping). sell. Finally, the primer and probe sets of the present invention provide high throughput and efficient processing.

A. Definitions As used herein, the singular forms include the plural correspondence unless the context contradicts. Regarding the recitation of numerical ranges herein, each intervening number is clearly included with the same degree of accuracy. For example, the range 6-9 includes the numbers 7 and 8 in addition to 6 and 9, and the range 6.0-7.0 includes 6.0, 6.1, 6.2. , 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0 are expressly included.

a) Amplicon As used herein, the term “amplicon” means the product of a natural or artificial amplification reaction. Examples of amplicons include PCR, real-time PCR, RT-PCR, competitive RT-PCR, ligase chain reaction (LCR), gap LCR, strand displacement amplification (SDA), nucleic acid sequence based ) DNA or RNA products (usually genes, DNA or RNA segments) produced by amplification (NASBA), transcription-mediated amplification (TMA), and the like.

b) Amplification, amplification method or amplification reaction As used herein, “amplification”, “amplification method” or “amplification reaction” are used interchangeably herein to refer to specific nucleic acids (all types) in a test sample. A method or process for increasing the amount of display of a population of sequences (eg, target sequence or target nucleic acid). Specific examples of amplification methods that can be used in the present invention include PCR, real-time PCR, RT-PCR, competitive RT-PCR, LCR, gap LCR, SDA, NASBA, TMA, etc. (all of which are known to those skilled in the art) .), But is not limited thereto.

c) Amplification conditions The term “amplification conditions” as used herein refers to conditions that promote primer sequence annealing and / or extension. Such conditions are well known to those skilled in the art and depend on the amplification method selected. For example, PCR amplification conditions generally include thermal cycling, such as cycling of the reaction mixture between two or more temperatures. In isothermal amplification reactions, an initial temperature increase may be required to initiate the reaction, but amplification occurs without thermal cycling. Amplification conditions include all reaction conditions including, but not limited to, temperature and temperature cycling, buffers, salts, ionic strength, pH, and the like.

d) Amplification reagent As used herein, the term “amplification reagent” refers to a reagent used in an amplification reaction, and includes a buffer, a reagent, reverse transcription and / or an enzyme having polymerase activity or exonuclease activity; Factors such as magnesium or manganese, salts; and deoxynucleotide triphosphates (dNTPs), such as deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate Phosphate (dTTP) and deoxyuridine triphosphate (dUTP) may be included (but not limited to). Amplification reagents can be easily selected by those skilled in the art depending on the amplification method used.

e) Directly detectable and indirectly detectable The term “directly detectable” as used herein with reference to a detectable label or detectable moiety means that the detectable label or detectable moiety is , Meaning that no further reaction or manipulation is required to be detectable. For example, the fluorescent moiety can be detected directly by fluorescence spectroscopy. In contrast, the term “indirectly detectable” when used herein with reference to a detectable label or detectable moiety means that the detectable label or detectable moiety has not been further reacted or manipulated. Means that it can be detected. For example, a hapten can be detected after reaction with an appropriate antibody coupled to a reporter such as a fluorescent dye.

f) Fluorophore, fluorescent moiety, fluorescent label or fluorescent dye The terms “fluorophore”, “fluorescent moiety”, “fluorescent label” and “fluorescent dye” are used interchangeably herein and A molecule that absorbs a quantum of electromagnetic radiation of a wavelength and emits one or more photons of different wavelengths, typically longer wavelengths, in response. A large number of fluorescent dyes of a wide variety of structures and features are suitable for use in the practice of the present invention. Methods and materials for fluorescent labeling of nucleic acid molecules are known (RP Haugland, “Molecular Probes: Handbook of Fluorescent Probes and Research Chemicals 1992-1994”, 5th Ed., 1994, Molecular Incorporated. Please refer.) Preferably, the fluorescent label or moiety absorbs and emits light with high efficiency (eg, exhibits a high molar absorption coefficient and high fluorescence quantum yield at the excitation wavelength used) and exhibits light stability (eg, performs analysis). It does not undergo significant degradation upon photoexcitation within the time required for the Instead of being directly detectable per se, some fluorescent dyes transfer energy to another fluorescent dye in a process called fluorescence resonance energy transfer (FRET), where the second dye Generate a detection signal. Such FRET fluorescent dye pairs are also included in the term “fluorescent moiety”. Physically linked fluorescent reporter / quencher moieties are also within the scope of the present invention. In these embodiments, the quencher moiety prevents detection of a fluorescent signal from the reporter moiety when the fluorescent reporter and quencher moiety are maintained in proximity, eg, at the end of a probe. When these two parts are physically separated, for example after cleavage with DNA polymerase, the fluorescent signal from the reporter moiety becomes detectable.

g) Hybridization As used herein, the term “hybridization” refers to nucleic acid sequences that are sufficiently complementary to form a complex by Watson-Crick or non-canonical base pairing. The formation of a complex. For example, when a primer “hybridizes” to a target sequence (template), such a complex (or hybrid) is sufficient to perform the priming function required by, for example, a DNA polymerase to initiate DNA synthesis. It is as stable as possible. It will be appreciated by those skilled in the art that the hybridizing sequences need not have perfect complementarity to form a stable hybrid. In many cases, stable hybrids will occur when less than about 10% of the bases are mismatched. Thus, as used herein, the term “complementary” refers to oligonucleotides that form stable duplexes with the corresponding complement under assay conditions, generally about 80%, about 81%, about 82. %, About 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, It means when there is about 95%, about 96%, about 97%, about 98%, or about 99% or more homology. A method for estimating and adjusting the stringency of hybridization conditions such that at least sequences with a desired level of complementarity stably hybridize, while sequences with lower complementarity do not stably hybridize, is It is understood by the contractor. Specific examples of hybridization conditions and parameters are described in, for example, Sambrook et al., “Molecular Cloning: A Laboratory Manual”, 1989, Second Edition, Cold Spring Harbor Press: Plainview, NY; M.M. Ausubel, “Current Protocols in Molecular Biology”, 1994, John Wiley & Sons: Secaucus, NJ.

h) Labeled or labeled with a detectable label
As used herein, the terms “labeled” and “labeled with a detectable label (or substance or moiety)” are used interchangeably herein and include an object (eg, a primer or Means that the probe can be visualized, for example after binding to another object (eg amplification product or amplicon). Preferably, the detectable label is selected such that it generates a signal that can be measured and whose intensity is related (eg, proportional) to the amount of bound object. A wide variety of systems for labeling and / or detecting nucleic acid molecules (eg, primers and probes) are well known in the art. Labeled nucleic acid can be incorporated into or onto a label that is detectable directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical or other means. Can be prepared by conjugation. Suitable detectable substances include, but are not limited to, radionuclides, fluorophores, chemiluminescent materials, microparticles, enzymes, colorimetric labels, magnetic labels, haptens, molecular beacons, aptamer beacons, and the like. Is not to be done.

i) Primer The term “primer” is used in the presence of nucleotides and substances for nucleic acid polymerization (eg, DNA-dependent or RNA-dependent polymerase), under appropriate amplification conditions (eg, buffer, salt, temperature and pH). Means an oligonucleotide that can act as a starting point for the synthesis of a primer extension product that is the complementary strand of a nucleic acid (all types of DNA or RNA). The primer can be single-stranded or double-stranded. If double stranded, the primer can be first treated (eg, denatured) to allow for separation of the strand before being used to prepare extension products. Such denaturing steps are typically performed using heat, but may alternatively be performed by the use of alkali and subsequent neutralization. The primers of the present invention have a length of about 15-50 nucleotides, preferably about 20 to about 40 nucleotides, and most preferably about 22-30 nucleotides. The primers of the present invention may contain additional nucleotides in addition to those described in more detail herein. For example, primers used in SDA can include a restriction endonuclease recognition site 5 ′ to the target binding sequence (see US Pat. Nos. 5,270,184 and 5,455,166). Note) NASBA and TMA primers can include an RNA polymerase promoter linked to the target binding sequence of the primer. Methods for linking such specialized sequences to target binding sequences used in selected amplification reactions are well known to those skilled in the art. In some cases, the primer can also be labeled with a detectable label.

  The term “forward primer” refers to a primer that hybridizes (or anneals) to a target sequence (eg, a template strand). The term “reverse primer” refers to a primer that hybridizes (or anneals) to the complementary strand of a target sequence. The forward primer hybridizes to the target sequence 5 'to the reverse primer.

j) Primer set As used herein, the term “primer set” refers to two or more primers that can together prime the amplification of a target sequence or target nucleic acid of interest (eg, a target sequence in HPV). Means. In certain embodiments, the term “primer set” refers to a 5 ′ (upstream) primer (or forward primer) that hybridizes to the 5 ′ end of a target sequence or target nucleic acid to be amplified, and a target sequence or target to be amplified. A pair of primers is meant, including a 3 ′ (downstream) primer (or reverse primer) that hybridizes to the complement of nucleic acids. Such primer sets or primer pairs are particularly useful in PCR amplification reactions.

k) Probe As used herein, the term “probe” refers to an oligonucleotide that can selectively hybridize to at least a portion of a target sequence (eg, a portion of the amplified target sequence) under suitable amplification conditions. Means. In general, probe sequences are recognized as either “complementary” (ie, complementary to the code or sense strand (+)) or “reversely complementary” (ie, complementary to the antisense strand (−)). It is done. The probes of the present invention have a length of about 10-50 nucleotides, preferably about 12-35 nucleotides, most preferably 14-25 nucleotides. In some cases, the probe can be labeled with a detectable label.

l) Primer and probe set As used herein, the term “primer and probe set” is used to detect two or more primers that can be primed together for amplification of a target sequence or target nucleic acid and a target sequence or target nucleic acid. It means a combination comprising at least one probe that can. The probe generally hybridizes to a strand of amplification product (or amplicon) to form an amplification product / probe hybrid, which can be detected using conventional techniques known to those skilled in the art.

m) Target Sequence or Target Nucleic Acid The terms “target sequence” and “target nucleic acid” are used interchangeably herein and mean those whose presence or absence is desired to be detected. In the context of the present invention, the target sequence preferably comprises a nucleic acid sequence in which one or more primers form a complex. The target sequence may also include a region that hybridizes to the probe, where the probe forms a stable hybrid under appropriate amplification conditions. As will be appreciated by those skilled in the art, the target sequence can be single-stranded or double-stranded. In the context of the present invention, the target sequence of interest is located in the L1 region of HPV or in the open reading frame of the human beta globin gene.

n) Test Sample As used herein, the term “test sample” generally refers to the analyte of interest, eg HPV, in particular HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56. , 58, 59, 66 and 68, biological materials suspected and / or tested to contain human beta globin sequences or combinations thereof. The test sample can be of any biological origin, such as cervical, vaginal or anal swab or brush, or physiological fluids such as whole blood, serum, plasma, interstitial fluid, saliva, ophthalmic lens fluid, cerebrospinal fluid. , Sweat, urine, milk, ascites, mucus, runny nose, sputum, synovial fluid, peritoneal fluid, vaginal fluid, menstruation, amniotic fluid, semen, and the like (but not limited to). The test sample can be used directly as obtained from biological sources or after pretreatment to modify the properties of the sample. For example, such pretreatment can include preparing plasma from blood, diluting mucus, and the like. Pretreatment methods can also include filtration, precipitation, dilution, distillation, mixing, concentration, inactivation of interfering components, addition of reagents, cell lysis, and the like. In addition, it may be beneficial to modify the solid test sample to form a liquid medium or to liberate the analyte.

B. Primers, probes and primer and probe sets In one embodiment, the present invention relates to human papillomavirus (HPV) 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, in a test sample. It relates to one or more primers for amplifying types 59, 66 and 68. Such one or more primers may comprise any of the sequences shown in Table A below, the complement of any of the sequences shown in Table A below, and the combinations shown in Table A below and / or their complements. A primer having a sequence comprising or consisting of any of the above may be included.

In one embodiment, the present invention relates to HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, in a test sample containing one or more of the primers listed in Table A. The present invention relates to a primer set for amplifying types 58, 59, 66 and 68. In particular, the primer set may include:
(A) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, their complements (for example, one or more of SEQ ID NO: 1, SEQ ID NO: 2 or the complement of SEQ ID NO: 3) and any combination thereof At least one forward primer having a sequence selected from: (b) SEQ ID NO: 4, SEQ ID NO: 5, their complements (eg, one or more of SEQ ID NO: 4 or the complement of SEQ ID NO: 5) and any of them At least one reverse primer having a sequence selected from the group consisting of:

  In another embodiment, the present invention is for detecting HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 in a test sample. It relates to one or more probes. Such one or more probes may comprise any of the sequences shown in Table B below, the complement of any of the sequences shown in Table B below, and the combinations shown in Table B below and / or their complements. A probe having a sequence comprising or consisting of any of the above may be included. For example, such one or more probes may be a single probe listed in Table B below, or a single complement of one of the probes listed in Table B below (eg, (a) SEQ ID NO: 8, (b) complement of SEQ ID NO: 10, (c) SEQ ID NO: 12 or (d) SEQ ID NO: 21), all of the probes listed in Table B below (SEQ ID NOs: 8-21), The complements of all probes listed in Table B (complements SEQ ID NOs: 8-21), or probes listed in Table B below and / or probes listed in Table B below. Any combination of complements (eg, (a) SEQ ID NO: 10 and SEQ ID NO: 16; (b) the complement of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 15 and SEQ ID NO: 21; (c) Complement of SEQ ID NO: 8, arrangement The complement of SEQ ID NO: 9 and SEQ ID NO: 16; (d) The complement of SEQ ID NO: 11, SEQ ID NO: 17, SEQ ID NO: 18 and the complement of SEQ ID NO: 20; (e) SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 12 Complement, the complement of SEQ ID NO: 14 and SEQ ID NO: 20; or (f) SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20).

In another embodiment, the present invention relates to HPV 16, 18 in a test sample containing one or more of the primers listed in Table A above and one or more of the probes listed in Table B above. , 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. For example, the primer and probe set can include:
(A) at least one forward primer having the sequence of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 or their complements, and at least one reverse having the sequence of SEQ ID NO: 4 and SEQ ID NO: 5 or their complements And (b) SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, At least one probe having the sequence of SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21 or their complements.

Preferably, the primer and probe set are
(A) three forward primers having the sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 or their complements, and two reverses having the sequences of SEQ ID NO: 4 and SEQ ID NO: 5 or their complements A primer,
(B) SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 , And 14 probes having the sequences of SEQ ID NO: 20 and SEQ ID NO: 21 or their complements.

  In yet another embodiment, the present invention relates to primers for amplifying human beta globin sequences in a test sample. Such one or more primers include any of the sequences shown in Table C below, the complement of any of the sequences shown in Table C below, and combinations of the sequences shown in Table C below and / or their complements. A primer having a sequence comprising or consisting of any of the above.

  In yet another embodiment, the present invention relates to a probe for detecting human beta globin sequences in a test sample. The probe has a sequence comprising or consisting of actggggcatg tggagacaga (SEQ ID NO: 22) or its complement.

  In yet another embodiment, the invention comprises in a test sample comprising at least two primers selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, the complement of SEQ ID NO: 6 and the complement of SEQ ID NO: 7. Relates to a primer set for amplifying the endogenous human beta globin. Preferably, the primer set comprises SEQ ID NO: 6 and SEQ ID NO: 7.

In yet another embodiment, the present invention provides a test sample comprising one or more of the primers listed in Table C above or their complements and a probe having the sequence of SEQ ID NO: 22 or its complement. It relates to a primer and probe set for detecting endogenous human beta globin. For example, the primer and probe set is
(A) at least one primer having the sequence of SEQ ID NO: 6, SEQ ID NO: 7, the complement of SEQ ID NO: 6 or the complement of SEQ ID NO: 7,
(B) a probe having the sequence of SEQ ID NO: 22 or its complement.

Preferably, the primer and probe set are
(A) a forward primer having the sequence of SEQ ID NO: 6 or its complement, and a reverse primer having the sequence of SEQ ID NO: 7 or its complement;
(B) a probe having the sequence of SEQ ID NO: 22 or its complement.

  The primers and probes described above for amplifying and detecting human beta globin in a test sample can be used to obtain an internal control (IC) amplicon in the HPV assay. Detection of human beta globin in the HPV assay serves as a sample validity control for cell validity, sample extraction and amplification effectiveness. More specifically, this internal control serves to prove that each test sample has sufficient cell input for accurate HPV detection and is properly processed, and whether there are any inhibitors of amplification. Indicates whether or not

  One or more oligonucleotide analogues can be produced based on the primers and probes of the invention. Such analogs may contain alternative structures, such as peptide nucleic acids, ie “PNA” (eg, molecules having a peptide-like backbone instead of the phosphate sugar backbone of a naturally occurring nucleic acid). These alternative structures are also included in the present invention. Similarly, it is understood that the primers and probes of the present invention may contain nucleobase deletions, additions and / or substitutions. However, such modifications should not negatively affect the properties of these sequences. In particular, the modification should not result in a significant reduction in the hybridizing properties of the primers and probes herein.

  The primers and probes of the present invention can be produced by any of a variety of methods known in the art (eg, Sambrook et al., “Molecular Cloning. A Laboratory Manual”, 1989, 2. Supp. Ed., Cold Spring). Harbor Laboratory Press: New York, NY; “PCR Protocols. A Guide to Methods and Applications”, 1990, M. A. Innis (ed), Academic Press: N iTiTi, Y. Laboratory Technologies in Bioc “History and Molecular Biology (Parts I and II)”, 1993, Elsevier Science; “PCR Strategies”, 1995, MA Innis (ed.), Academic Press: New Yol, Nol Y; 2002, FM Ausubel (eds.), 5. Supp. Ed., John Wiley & Sons: Secaucus, NJ). For example, the primers and probes herein can be synthesized by chemical synthesis and template-based polymerization (eg, Narang et al., Meth. Enzymol., 1979, 68: 90-98; Brown et al., Meth. Enzymol., 1979, 68: 109-151 and Belousov et al., Nucleic Acids Res., 1997, 25: 3440-3444).

  For example, primers and probes can be produced using an automated solid phase method based on the phosphoramidite method. In such a method, each nucleotide is added individually to the 5 'end of the growing oligonucleotide chain that is attached to the solid support at the 3' end. The additional nucleotide is in the form of a trivalent 3'-phosphoramidite protected at the 5 'position by a dimethoxytrityl (or DMT) group. After base-induced phosphoramidite coupling, mild oxidation and DMT removal giving a pentavalent phosphotriester intermediate provides a new site for oligonucleotide extension. The primer or probe is then cleaved from the solid support and the phosphodiester and exocyclic amino group are deprotected with ammonium hydroxide. These syntheses are performed by oligo synthesizers such as Perkin Elmer / Applied Biosystems, Inc. (Foster City, CA), DuPont (Wilmington, DE), or commercially available from Milligen (Bedford, Mass.). Alternatively, the primers and probes of the present invention are described in, for example, Midland Certified Reagent Company (Midland, TX), ExpressGen, Inc. (Chicago, IL), Operon Technologies, Inc. (Huntsville, AL), BioSearch Technologies, Inc. (Novato, CA) and many others can be specially manufactured and ordered from various commercial sources well known in the art.

  Purification of the primers and probes of the invention can be performed by any of a variety of methods well known in the art, if necessary or desired. Purification of primers and probes can be performed using natural acrylamide gel electrophoresis, eg, Pearson et al., J. MoI. Chrom. , 1983, 255: 137-149 (see McFarland et al., Nucleic Acids Res., 1979, 7: 1067-1080).

  The primer and probe sequences are described in chemical degradation (see Maxam et al., Methods of Enzymology, 1980, 65: 499-560), matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (Pieles et al., Nucleic Acids Res., 1993, 21: 3191-3196) (but not limited to), mass spectrometry after a combination of alkaline phosphatase and exonuclease digestion (Wu et al. Anal. Biochem. , 2001, 290: 347-352) and the like can be confirmed using any suitable sequencing method known in the art.

  As already described above, modified primers and probes can be produced using any of several means known in the art. Non-limiting examples of such modifications include methylation, “caps”, one or more substitutions of natural nucleotides with analogs, and internucleotide modifications, such as those having uncharged linkages (eg, Methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) or charged linkages (eg phosphorothioates, phosphorodithioates, etc.). Primers and probes may include one or more additional covalent moieties, such as proteins (eg, nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (eg, acridine, psoralen, etc.), chelators ( For example, it may contain a metal, a radioactive metal, an oxidizable metal, etc.) and an alkylator. Primers and probes can be derivatized by formation of methyl or ethyl phosphotriester or alkyl phosphoramidate linkages. Furthermore, the primers, probes or primers and probes of the invention can also be modified with a detectable label.

  As mentioned above, in certain embodiments of the invention, the primer, probe or both the primer and probe are labeled with a detectable label or moiety prior to use in an amplification / detection method. sell. Preferably, as used in the methods herein, one or more probes are labeled with a detectable label or moiety. The role of the detectable label is to allow visualization and detection of the amplified target sequence (eg, amplicon). Preferably, the detectable label is selected such that it produces a signal that can be measured and whose intensity is related (eg, proportional) to the amount of amplification product in the test sample being analyzed. The

  The linkage between the one or more probes and the detectable label can be covalent or non-covalent. Labeled probes can be produced by incorporation of a detectable moiety or binding to a detectable moiety. The label can be attached directly or indirectly (eg, via a linker) to the nucleic acid sequence. Various length linkers or spacer arms are known in the art and are commercially available to reduce steric hindrance or impart other useful or desired properties to the resulting labeled molecule. (See, for example, Mansfield et al., Mol. Cell. Probes, 1995, 9: 145-156).

  Methods for labeling oligonucleotides such as probes are known to those skilled in the art. For a review of labeling protocols and label detection techniques, see for example L. J. et al. Kricka, Ann. Clin. Biochem. , 2002, 39: 114-129; van Gijswijk et al., Expert Rev. Mol. Diagn. , 2001, 1: 81-91; and Joos et al., J Biotechnol, 1994, 35: 135-153. Standard nucleic acid labeling methods include radioactive material incorporation, fluorescent dyes (see Smith et al., Nucl. Acids Res., 1985, 13: 2399-2412) or enzymes (Connolly et al., Nucl. Acids. Res. , 1985, 13: 4485-4502); chemical modification of the nucleic acid molecule to make it detectable immunochemically or by other affinity reactions (Broker et al., Nucl. Acids Res., 1978, 5: 363-384; Bayer et al., Methods of Biochem.Analysis, 1980, 26: 1-45; Langer et al., Proc. Natl. Acad. Sci USA, 1981, 78: 6633-6737; Et , Nucl.Acids Res., 1983, 11: 6167-6184; Brigati et al., Virol., 1983, 126: 32-50; Tchen et al., Proc. Natl.Acad.Sci.USA, 1984, 81: 3466-3470; See Landegent et al., Exp. Cell Res., 1984, 15: 61-72; and AH Hopman et al., Exp. Cell Res., 1987, 169: 357-368.); For example, random priming, nick translation, PCR, and tailing with terminal transferase (for a review on enzyme labeling see, eg, Temsumani et al., Mol. Biotechnol., 1996, 5: 223-232. There.) Are included.

Any of a wide variety of detectable labels can be used in the present invention. Suitable detectable labels include various ligands, radionuclides (eg, ³² P, ³⁵ S, ³ H, ¹⁴ C, ¹²⁵ I, ¹³¹ I, etc.); fluorescent dyes; chemiluminescent substances (eg, acridinium esters Spectroscopically separable inorganic fluorescent semiconductor nanocrystals (eg quantum dots), metal nanoparticles (eg gold, silver, copper and platinum) or nanoclusters; enzymes (eg horseradish peroxidase) , Beta-galactosidase, luciferase, alkaline phosphatase); colorimetric labels (eg, dyes, colloidal gold, etc.); magnetic labels (eg, Dynabeads ™); and biotin, dioxygenin or other haptens (limited to these) Not antisera or monoclonal) Protein for the body is available.

  In certain embodiments, the detection probes of the invention are fluorescently labeled. Numerous known fluorescent labeling moieties with a wide variety of chemical structures and physical properties are suitable for use in the practice of the present invention. Suitable fluorescent dyes include QUASAR® dyes available from Biosearch Technologies, Novato, CA, fluorescein and fluorescein dyes such as fluorescein isothiocyanin or FITC, naphthofluorescein, 4 ′, 5′-dichloro-2 ′. , 7'-dimethoxy-fluorescein, 6-carboxyfluorescein (eg FAM), VIC, NED, carbocyanine, merocyanine, cytilyl dye, oxonol dye, fucoerythrin, erythrosine, eosin, rhodamine dye (eg carboxytetramethylrhodamine or TAMRA , Carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), lysamine rhodamine B, rhodamine 6G, rhodamine green Rhodamine red, tetramethylrhodamine or TMR), coumarin and coumarin dyes (eg methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin and aminomethylcoumarin or AMCA), Oregon green dyes (eg Oregon Green 488, Oregon Green 500, Oregon Green) 514), Texas Red, Texas Red-X, Spectrum Red ™, Spectrum Green ™, cyanine dyes (eg, Cy-3 ™, Cy-5 ™, Cy-3.5 ™) , Cy-5.5 ™), Alexa Fluor dyes (eg, Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, Al exa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660 and Alexa Fluor 680, BODIPY dyes (e.g., BODIPY FL, BODIPY R6G, BODIPY 50R) 568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665), IRDyes (eg, IRD 40, IRD 700, IRD 800) and the like. Is not to be done. Specific examples of other suitable fluorescent dyes that can be used and methods for linking or incorporating fluorescent dyes into oligonucleotides such as probes can be found in RP Haugland, “The Handbook of Fluorescent Probes and Research Chemicals,” Publisher, Molecular Probes, Inc. Eugene, Oreg. (June 1992). Fluorescent dyes and labeling kits are described in, for example, Amersham Biosciences, Inc. (Piscataway, NJ), Molecular Probes Inc. (Eugene, OR) and New England Biolabs Inc. (Beverly, MA).

  Some fluorescent groups (donors) transfer energy to another fluorescent group (acceptor) in the course of fluorescence resonance energy transfer (FRET) instead of being directly detectable themselves, and the second The group produces a detectable fluorescent signal. In these embodiments, the probe can be detectable, for example, when hybridized to an amplified target sequence. Specific examples of FRET acceptor / donor pairs suitable for use in the present invention include, for example, fluorescein / tetramethylrhodamine, IAEDANS / FITC, IAEDANS / 5- (iodoacetamido) fluorescein, B-phycoerythrin / Cy-5 and EDANS / Dabcyl is included, but is not limited thereto.

  The use of physically linked fluorescent reporter / quencher molecules is also within the scope of the present invention. In TaqMan® assays (eg, as described in US Pat. Nos. 5,210,015, 5,804,375, 5,487,792 and 6,214,979) Or as Molecular Beacons (eg, Tyagi et al., Nature Biotechnol., 1996, 14: 303-308; Tyagi et al., Nature Biotechnol., 1998, 16: 49-53; Kostrikis et al., 1998, 1998). Sokol et al., Proc. Natl. Acad. Sci USA, 1998, 95: 11538-11543; Marras et al., Genet. Anal, 1999, 14: 151-1. 6; and US Pat. Nos. 5,846,726, 5,925,517, 6,277,581 and 6,235,504) use of such systems is Well known to those skilled in the art. In the TaqMan® assay format, the product of the amplification reaction can be detected as it is formed in a “real time” manner. As a result, amplification product / probe hybrids are formed and detected while the reaction mixture is under amplification conditions.

  In some embodiments of the invention, the PCR detection probe is a TaqMan®-like probe labeled with a fluorescent moiety at the 5 'end and a quenching moiety at the 3' end. Suitable fluorophores and quenchers for use with TaqMan®-like probes are US Pat. Nos. 5,210,015, 5,804,375, 5,487,792 and 6,214. , 979 and WO 01/86001, each of which is incorporated herein by reference. Specific examples of quenchers include DABCYL (eg, 4- (4′-dimethylaminophenylazo) benzoic acid) succinimidyl ester, diarylrhodamine carboxylic acid, succinimidyl ester (or QSY-7) and 4 ′, 5'-dinitrofluorescein carboxylic acid, succinimidyl ester (or QSY-33), all of which are available from Molecular Probes (which is part of Invitrogen, Carlsbad, CA), quencher. 1 (Q1; available from Epoch Biosciences, Bothell, WA), or “Black hole quencher” BHQ-1, BHQ-2 and BHQ-3 (BioSearch Tech) nogologies, Inc., available from Novato, Calif.) In certain embodiments, the PCR detection probe is FAM at the 5 ′ end and at the 3 ′ end. TaqMan®-like probe labeled with Black Hole Quencher® or Black Hole Quencher® plus (both available from Biosearch Technologies, Novato, Calif.).

  Also, a “tail” of normal or modified nucleotides can be added to the probe for detection purposes. A second hybridization with a nucleic acid that is complementary to the tail and contains one or more detectable labels (eg, fluorophore, enzyme, or radiolabeled base) is a visualization of the amplicon / probe hybrid. Enable.

  The choice of the particular labeling technique depends on the situation, eg the ease and cost of the labeling method, the spectral spacing between the different detectable labels used, the quality of the desired sample label, the hybridization reaction (eg hybridization Depends on several factors such as the effect of the detectable moiety (on the speed and / or efficiency of the process), the nature of the amplification method used, the nature of the detection system, the nature and intensity of the signal produced by the detectable label, etc. I will.

C. Amplification method The present invention for amplifying HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 target sequences and human beta globin target sequences in test samples The use of this primer or primer set is not limited to any particular nucleic acid amplification technique or any particular variation thereof. In fact, the primers and primer sets of the present invention can be prepared using various nucleic acid amplification methods known in the art (eg, Kimmel et al., Methods Enzymol., 1987, 152: 307-316; Sambrook et al., “Molecular Cloning. A Laboratory Manual). “, 1989, 2. Supp. Ed., Cold Spring Harbor Laboratory Press: New York, NY;” “Short Protocols in Molecular Biology”, FM Ausubel (ed.), 2002. e. & Sons: Secaucus, NJ).

  Such nucleic acid amplification methods include, but are not limited to, polymerase chain reaction (PCR). PCR is described in “PCR Protocols: A Guide to Methods and Applications”, M.M. A. Innis (ed.), 1990, Academic Press: New York; “PCR Strategies”, M.M. A. Innis (ed.), 1995, Academic Press: New York; “Polymerase chain reaction: basic principals and automation in PCR. A Practical Approch, Nr, et al. (1998); And U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,889,818, which are not limited thereto (each of which is incorporated by reference in its entirety). In a number of references such as). TaqMan®-based assays (see Holland et al., Proc. Natl. Acad. Sci, 1991, 88: 7276-7280) and reverse transcription polymerase chain reaction (or RT-PCR; see, eg, US Pat. No. 5 , 322, 770 and 5,310, 652 (each of which is incorporated herein by reference) are also included.

  In general, in PCR, primer pairs are added in excess to a test sample obtained from a subject (and thus in contact with the test sample) and hybridized to the complementary strand of the target nucleic acid. Each of the primers is extended with DNA polymerase using the target sequence as a template. The extension product becomes the target itself after dissociation (denaturation) from the original target strand. New primers are then hybridized, extended with the polymerase, and the cycle repeated to increase the number of amplicon copies exponentially. Specific examples of DNA polymerases that can produce primer extension products in a PCR reaction include E. coli DNA polymerase I, Klenow fragment of DNA polymerase I, T4 DNA polymerase, various sources (eg, Perkin Elmer, Thermostable DNA polymerase, Thermus thermophilus (USB Corporation, Cleveland, OH), Bacillus stereo, isolated from Thermus aquaticus (Taq), available from Waltham, MA Thermophilus (Bio-Rad Laboratories, Hercul) s, CA), AmpliTaq Gold (R) Enzyme (Applied Biosystems, Foster City, CA), Recombinant Thermus thermophilus (rTth) DNA polymerase (Applied Biosystems, Foster City, Foster City) (Thermococcus literalis) ("Vent" polymerase, New England Biolabs, Ipswich, MA). The RNA target sequence can be amplified by reverse transcription (RT) of the mRNA into cDNA followed by PCR (RT-PCR) (as described above). Alternatively, a single enzyme can be used for both steps as described in US Pat. No. 5,322,770, which is incorporated herein by reference.

  In addition to the enzymatic thermal amplification methods described above, isothermal enzymatic amplification reactions can be used to amplify HPV or beta globin target sequences using the primers and primer sets of the present invention (Andras et al., Mol. Biotechnol. 2001, 19: 29-44). These methods include Transcription-Mediated Amplification (TMA; TMA Kwoh et al., Proc. Natl. Acad. Sci. USA, 1989, 86: 1173-1177; Giacetti et al., J Clin. Microbiol., 2002, 40: 2408-2419; and US Pat. No. 5,399,491.); Self-Sustained Sequence Replication (3SR; 3SR is described in Guatelli et al., Proc. Natl. Acad.Sci.USA, 1990, 87: 1874-1848; and Fahy et al., PCR Methods and Applications, 19 1, 1: 25-33); Nucleic Acid Sequence Based Amplification (NASBA; NASBA is described in Kievits et al., J Virol. Methods, 1991, 35: 273-286; and US Pat. No. 5,130,238). ) And Strand Displacement Amplification (SDA; SDA is described in Walker et al., PNAS, 1992, 89: 392-396; EP 0 500 224 A2). It is not something.

  Strand displacement amplification involves the ability of a restriction endonuclease to break into the unmodified strand of its target DNA and the action of an exonuclease deficient DNA polymerase to extend the 3 ′ end of the break and remove the downstream DNA strand at a constant temperature. (See Walker et al., Proc. Natl. Acad. Sci. USA, 1992). Primers used in SDA include restriction endonuclease recognition at a site 5 ′ to the target binding sequence (US Pat. Nos. 5,270,184 and 5,344,166, each of which is incorporated herein by reference). ), Which is incorporated herein by reference).

  Nucleic Acid Sequence Based Amplification (NASBA) consists of three enzymes (e.g., RNase H, chicken myeloblastosis virus (AMV)) that act cooperatively at low isothermal temperatures, typically 41 ° C. ) Reverse transcriptase and T7 RNA polymerase) (see Compton, Nature, 1991, 350: 91-92; Chan et al., Rev. Med. Microbiol, 1999, 10: 185-196). The product of the NASBA reaction is primarily single stranded RNA.

  The Self Sustaining Sequence Replication (3SR) reaction is a very efficient method for isothermal amplification of target DNA or RNA sequences. The 3SR system includes the collective activity of AMV reverse transcriptase, E. coli RNase H and DNA-dependent RNA polymerase (eg, T7 RNA polymerase).

  Transcription-mediated amplification (TMA) consists of RNA polymerase to obtain RNA from a promoter engineered in the primer region, reverse transcriptase to obtain complementary DNA from an RNA template, and RNase to remove the RNA from cDNA (See Guatelli et al., Proc. Natl. Acad. Sci. USA, 1990).

  NASBA, 3SR and TMA primers require an RNA polymerase promoter linked to the target binding sequence of the primer. The promoter or promoter sequence for incorporation into the primer is a nucleic acid sequence that is specifically recognized by RNA polymerase (either naturally occurring, synthetically produced, or the product of restriction digests) Where the RNA polymerase recognizes and binds to the sequence and initiates the process of transcription, thereby giving an RNA transcript. Examples of useful promoters include those recognized by certain bacteriophage polymerases, such as those derived from bacteriophage T3, T7 or SP6, or promoters derived from E. coli.

D. Detection Methods In certain embodiments of the invention, the probes herein are used to detect amplification products generated by the amplification reaction. The probes herein can be used with various well-known uniform or non-uniform methods.

  A homogeneous detection method includes a FRET label that binds to the probe and emits a signal in the presence of a target sequence, Molecular Beacons (Tyagi et al., Nature Biotechnol., 1996, 14: 303-308; Tyagi; Nature Biotechnol, 1998, 16: 49-53; Kostrikis et al., Science, 1998, 279: 1228-1229; Sokol et al., Proc. Natl. Acad. Sci USA, 1998, 95: 11538-11543; Marras et al., Genet. Anal., 1999, 14: 151-156; and U.S. Patent Nos. 5,846,726, 5,925,517, 6,277,581 and 6,235. 504) and TaqMan® assay (US Pat. Nos. 5,210,015, 5,804,375, 5,487,792 and 6,214,979) and Use of WO 01/86001), but is not limited thereto. Using these detection techniques, the product of the amplification reaction can be detected as it is formed, i.e., in a real-time manner. As a result, amplification product / probe hybrids are formed and detected while the reaction mixture is under amplification conditions.

  In certain embodiments, the probes of the present invention are used in TaqMan® assays. In the TaqMan® assay, analysis is performed with thermal cycling by monitoring the generation of a fluorescent signal. The assay system has the ability to generate quantitative data that allows determination of target copy number. For example, a standard curve can be generated using a serial dilution of a pre-quantified suspension of one or more HPV types or human beta globin sequences, whereas an unknown sample can be compared. The TaqMan® assay uses, for example, an AmpliTaqGold ™ DNA polymerase with endogenous 5 ′ nuclease activity to digest a probe labeled with both a fluorescent reporter dye and a quenching moiety as described above. And it is done simply. Measure the change in fluorescence that occurs during the amplification cycle as the probe is digested, separate the fluorescent and quenching moieties, and obtain an assay result by causing an increase in fluorescent signal proportional to the amplification of the target sequence .

  Other examples of homogeneous detection methods include hybridization protection assays (HPA). In such assays, non-nucleotidic linker arm chemistry (see US Pat. Nos. 5,585,481 and 5,185,439) is used to use the highly chemiluminescent molecule acrizi. The probe is labeled with a nium ester (AE) (see Weeks et al., Clin. Chem., 1983, 29: 1474-1479; Berry et al., Clin. Chem., 1988, 34: 2087-2090). Chemiluminescence is triggered by hydrolysis of AE with alkaline hydrogen peroxide. In this case, the hydrolysis gives an excited N-methylacridone which is then deactivated with the emission of light. In the absence of the target sequence, AE hydrolysis is rapid. However, when the probe is bound to the target sequence, the rate of AE hydrolysis is significantly reduced. Therefore, hybridized AE-labeled probes and non-hybridized AE-labeled probes can be detected directly in solution without the need for physical separation.

  Heterogeneous detection systems are also well known in the art and generally use a capture material to separate amplified sequences from other materials in the reaction mixture. The capture material typically comprises a solid support material (eg, microtiter wells, beads, chips, etc.) coated with one or more specific binding sequences. The binding sequence can be complementary to the tail sequence added to the oligonucleotide probe of the present invention. Alternatively, the binding sequence can be complementary to the sequence of the capture oligonucleotide and can itself comprise a sequence that is complementary to the tail sequence of the probe. After separating the amplification product / probe hybrid bound to the capture material from the remaining reaction mixture, the amplification product / probe hybrid is detected using any detection method (eg, those described herein). Can be done.

E. Detection of HPV and human beta globin in a test sample In another embodiment, the present invention provides (a) HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56 in a test sample. , 58, 59, 66 and 68, (b) a method for detecting human beta globin sequences in a test sample, and (c) HPV 16, 18, 31 in a test sample 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 and methods for detecting the presence of human beta globin sequences.

  Typically, the method of the invention first involves obtaining a test sample from a subject. The subject from whom the test sample can be obtained is any mammal. Preferably, mammals include but are not limited to dogs, cats, rabbits, mice, rats, goats, sheep, cows, pigs, horses, non-human primates and humans. The test sample can be obtained from the subject using conventional techniques known to those skilled in the art. Preferably, the test sample is (i) at least one of HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68, and / or (ii) Contains or is suspected of containing at least one human beta globin sequence.

  After obtaining a test sample from a subject, contacting the test sample with a primer (and optionally one or more probes) from at least one of the primer sets or primer and probe sets disclosed herein, A reaction mixture is formed. The reaction mixture is then placed under amplification conditions. The primer hybridizes to any HPV nucleic acid and any human beta globin nucleic acid in the test sample. HPV or human globin nucleic acid present in the sample is amplified to obtain at least one amplification product (ie, at least one target sequence).

  By detecting hybridization between at least one amplification product and at least one of the probes of the invention (eg, one or more probes from the primers and probe sets herein), at least one amplification product Is detected. In particular, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, Detection of at least one amplification product with one or more of the probes having the sequence of SEQ ID NO: 20 and SEQ ID NO: 21 or their complements is performed in HPV 16, 18, 31, 33, 35, 39, 45, 51 in the test sample. , 52, 56, 58, 59, 66 and 68 are present. Detection of hybridization of the amplification product with a probe having the sequence of SEQ ID NO: 22 or its complement indicates the presence of a human beta globin sequence in the test sample. Preferably, the method of the invention comprises HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 and human beta globin sequences (as controls) in the test sample. Detecting at least one of those added. Most preferably, the detection of at least one HPV type and human beta globin sequence is performed simultaneously.

  The methods of the present invention can also be used to partially genotype (eg, identify) HPV types present and detected in a test sample. For example, to facilitate identification of different HPV types present in a test sample, each of the probes used in the methods herein can be labeled with a different detectable label that emits a different color of light. Preferably, however, for use in the methods of the invention, a probe having the sequence of SEQ ID NO: 8 is labeled with a first detectable label that emits a unique color (eg, red), and the sequence of SEQ ID NO: 9 is The probe having is similarly labeled with a second detectable label that emits a unique color (eg, green) and is different from the first detectable label. Sequence number 10, sequence number 11, sequence number 12, sequence number 13, sequence number 14, sequence number 15, sequence number 16, sequence number 17, sequence number 18, sequence number 19, sequence number 20 and sequence number 21 or those thereof If one or more of the probes having complementary sequences are also used in the method with SEQ ID NOs: 8 and 9, each of these probes could be labeled with the same detectable label (ie, each , Will be labeled with a third detectable label that emits a third characteristic color (eg, yellow)). Thus, the use of various types of labels with the HPV probes herein (see Table B) not only detects the presence of HPV in the test sample, but is also present in the sample. It is also possible to specifically identify the specific type to be. The detection and / or identification of HPV types 16 and 18 in the test sample (eg, by using probes having the sequences of SEQ ID NO: 8 and SEQ ID NO: 9) is useful and important. This is because these two HPV types cause at least 70% of cervical cancers.

F. In another embodiment of the kit , the present invention relates to HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68, according to the methods herein. And kits containing materials and reagents useful for the detection of human beta globin sequences. The kit can be used by diagnostic laboratories, laboratory laboratories or practitioners. In certain embodiments, the kit comprises at least one of the primer set or primer and probe set in Section B herein, and optionally an amplification reagent. Each kit preferably includes amplification reagents for a particular amplification method. Thus, a kit adapted for use with NASBA preferably contains a primer with an RNA polymerase promoter linked to a target binding sequence, while a kit adapted for use with SDA preferably comprises: Contains a primer containing a restriction endonuclease recognition site 5 'to the target binding sequence. Similarly, when the kit is adapted for use in a 5 ′ nuclease assay, such as the TaqMan® assay, a probe of the invention may contain at least one fluorescent reporter moiety and at least one quencher moiety.

  Suitable amplification reagents further include, for example, buffers, reagents, reverse transcriptase and / or enzymes having polymerase or exonuclease activity, enzyme cofactors such as magnesium or manganese; salts; deoxys suitable for carrying out the amplification reaction Contains one or more of nucleotide triphosphates (dNTPs). For example, a kit adapted for use with NASBA may contain appropriate amounts of reverse transcriptase, RNase H and T7 RNA polymerase. In a kit adapted for a transcription amplification reaction such as NASBA, for example, a buffer containing DMSO known to enhance the amplification reaction may be included.

  Depending on the method, the kit may further comprise one or more of a wash buffer, a hybridization buffer, a labeling buffer, a detection means and other reagents. The buffers and / or reagents are preferably optimized for the particular amplification / detection technique for which the kit is intended. Protocols for using these buffers and reagents for performing the various steps of the method can also be included in the kit.

  In addition, the kit may include an internal control for confirming cell validity, sample extraction, etc., to prevent the generation of false negative test results due to failure of the amplification, as a test standard for amplification efficiency. Is possible. The optimal internal control sequence is selected such that it does not compete with the target nucleic acid sequence in the amplification reaction. Preferably, the internal control is a beta globin primer (ie, at least one of SEQ ID NO: 6, SEQ ID NO: 7, the complement of SEQ ID NO: 6, or the complement of SEQ ID NO: 7) as previously described herein in Section B. And a probe (ie, SEQ ID NO: 22 or a complement of SEQ ID NO: 22).

  The kit may also contain reagents for isolation of nucleic acids from test samples prior to amplification prior to nucleic acid extraction.

  The reagent can be supplied in solid (eg, lyophilized) or liquid form. The kits of the present invention can optionally include various containers (eg, vials, ampoules, test tubes, flasks or bottles) for each individual buffer and / or reagent. Each component will generally be suitable that is aliquoted in its respective container or provided in a concentrated form. Other containers suitable for performing certain steps of the amplification / detection assay may also be provided. Individual containers are preferably kept sealed for commercial use.

  A kit was obtained for use with the amplification reagents and primer sets or primers and probes herein, instructions for processing test samples, extracting nucleic acid molecules, and / or performing tests, and obtained It may also contain instructions for interpreting the results, as well as notes in the form prescribed by government agencies. Such a description may in some cases be in printed form or other format of CD, DVD or recording medium.

  As a non-limiting illustration, a specific example of the present disclosure will be shown below.

Materials and Methods A. HPV and beta globin primer and probe design All oligonucleotides used in Examples 2-6 were synthesized using standard oligonucleotide synthesis methods known to those skilled in the art. All of the probes are single stranded oligonucleotides labeled using conventional techniques known in the art and having a fluorophore at the 5 'end and a quenching moiety at the 3' end. The 5 ′ label was used to label a VIC (this green-labeled dye was used to label a probe specific for HPV16 (ie, SEQ ID NO: 8; see Table B in Section B)), NED (this The yellow label was used to label a probe specific for HPV18 (ie, SEQ ID NO: 9; see Table B, Section B.)), FAM (this blue label dye is HPV 31, 33, Probes specific for 35, 39, 45, 51, 52, 56, 58, 59, 66 or 68 (eg, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 , SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21; see Table B of Section B))) or Weser ( (Quasar) (for human beta globin SEQ ID NO: 22). The 3 ′ label is a black hole quencher (BHQ), eg a probe specific to BHQ1-dT (HPV16, 31, 33, 35, 45, 51, 56, 59, 66 or 68 (section B). Used to label Table B.), BHQ2-dT (HPV18 (see Table B, Table B)) or a probe specific for human beta globin (SEQ ID NO: 22). Used to label), or BHQ1 plus (used to label probes specific to HPV39, 52 or 58 (see Table B in Section B)).

B. Real-time PCR
HPV DNA was extracted from the sample, concentrated and purified using magnetic particle technology. The technique captures nucleic acids and washes the particles to remove unbound sample components (see, eg, US Pat. No. 5,234,809). The bound nucleic acid was eluted and prepared for amplification. Three forward primers (ie, SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; see Table A in Section B) and two reverse primers (ie, sequences) that target the conserved L1 region The HPV primer mix of No. 4 and SEQ ID No. 5; see Table A in Section B.) was used to amplify the HPV target. These three forward primers (SEQ ID NOs: 1-3) and two reverse primers (SEQ ID NOs: 4-5) are collectively referred to herein as “HPV primer mixtures”. Signals for 14 human (HR) HPV genotypes (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) were sent to genotype specific probes ( That is, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21; see Table B in Section B.). All 14 of these probes are collectively referred to herein as “HPV probe sets”. Human beta globin target was amplified with a primer set (SEQ ID NO: 6 and 7) targeting the endogenous human beta globin sequence and detected with a beta globin probe (SEQ ID NO: 22). The PCR reaction consists of 14 units of AmpliTaq Gold enzyme, 7 mM magnesium chloride (as activation reagent) and other amplification reagents (0.6 mM dNTPs in Tris buffer, 73.5 nM ROX reference dye) in addition to the primers and probes. Containing).).

  Real-time amplification / detection was performed on an Abbott m2000rt instrument (Abbott Molecular Inc., Des Plaines, IL) using the following cycling conditions: one cycle at 92 ° C for 10 minutes; at 91 ° C for 30 seconds and at 54 ° C 4 cycles of 30 seconds; 38 cycles of 91 ° C. for 30 seconds, 52 ° C. for 30 seconds (with 1 second automatic extension per cycle) and 50 ° C. for 40 seconds. Fluorescence measurements were recorded during the 38 cycle reading process (50 ° C.).

Genotypic inclusion and partial genotyping In this example, 51 samples containing HPV DNA targets from each of the 14 genotypes were individually and combined to produce the HPV primer mixture of Example 1. And tested using the HPV probe set. Real-time PCR was performed as described in Example 1. Each HPV DNA target was tested at a concentration of 400,000 copies per reaction. As shown in Table 1 below, the results from the 51 samples have 14 samples with a single genotype, 25 samples with 2 genotypes, and 3 genotypes. Twelve samples were included. Moreover, as shown in Table 1, these results are shown accurately, and the presence or absence of HPV 16 and HPV 18 DNA was accurately determined in each case. This example shows that such a unique HPV primer mix and HPV probe set has 14 HR HPV genotypes (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 , 66 and 68), demonstrating that HPV16 and HPV18 can be distinguished from the other 12 HR HPV genotypes.

Probe Hybridization Specificity Two studies were performed to demonstrate the hybridization specificity of the HPV probe set described in Example 1. Using a single HR HPV probe (see Table 2) in the presence of individual HPV plasmid DNA targets at a concentration of about 10,000,000 copies per reaction, or a total of 14 HR HPV probes Real-time PCR was performed as described in Example 1 using the mixture containing (see Table 3). The results of those two studies shown in Tables 2 and 3 indicate the specificity of the selected HPV probes.

Detection of beta globin as a cell validity control In order to utilize human beta globin as a cell validity control, the beta globin signal is the same as in the cycle number (CN) obtained from analysis of the results of real-time PCR. Should be an indicator of the amount of cell input from clinical samples.

  Studies were conducted to evaluate the correlation between the amount of cell input from cultured HPV positive cell lines and the beta globin signal. The correlation between the beta globin signal and the cell input is shown in FIG. As demonstrated by previous CN, the study shows that increasing cell input concentration correlates with higher beta globin detection efficiency.

  In addition, another study was conducted to evaluate beta globin CN distribution in clinical samples. 1206 patient cervical samples collected in PreservCyt® solution (Cytyc Corporation, Marlborough, Mass.) Were analyzed and are shown in FIG. The 25%, 50% and 75% quartile values of the population have a beta globin CN of 21.29, 22.41 and 23.78, respectively, a minimum CN of 17.47 and a maximum of 36.01 Have CN. This study shows that beta globin can be used as a cell validity control.

  The human beta globin primer and probe described in Example 1 were used in real-time PCR to obtain the results shown in FIGS.

Analytical performance of primer and probe design This example shows the results of two studies. The first study was improved compared to the commonly used consensus GP5 + / GP6 + primer (GP5 + primer; TTTGTTACTGTGTAGATATACTAC-3 ′ (SEQ ID NO: 23), GP6 + primer: 5′-GAAAAAATAAAACTGTATAATCATATTC-3 ′ (SEQ ID NO: 24)) The analytical performance of the HPV primer mixture of Example 1 was shown. This study was performed using real-time PCR as described in Example 1. The performance of the unique HPV primer mixture of the present invention and the consensus GP5 + / GP6 + primer was evaluated using CN values. The results are shown in Table 4 and FIGS. An improvement in performance (CN improvement value> 1) was observed with 10/13 HPV genotypes (Table 4). A large improvement (CN improvement value> 10) was observed with HPV genotypes 39, 51, 52 and 68 and is shown in FIGS.

  In the second study, some of the unique probes of the invention specific for HPV types 16, 18, 31, 35, 39, 45, 51, 52, 58 and 59 (SEQ ID NOs: 8-10, 12-16). And 18-19) were examined in comparison to the probe sequence for the same HPV type disclosed in US Pat. No. 6,265,154B1 (see Table 5). The probe of US Pat. No. 6,265,154 was labeled as described in Example 1 to allow direct comparison when tested in real-time PCR. The performance was evaluated using CN value and fluorescence signal. The results are shown in Tables 6 and 7 and FIGS. Performance improvements (CN improvement value> 1 and fluorescence signal> 0.1) were observed with 5/10 HPV genotypes (Tables 6 and 7). Significant improvements in CN and fluorescence signals were observed with HPV genotypes 16, 18, 31, 52 and 59 and are shown in FIGS.

Clinical Performance of Primers and Probes of the Invention The sensitivity and specificity of the assay using the HPV primer mixture and HPV probe set described in Example 1 for the detection of HR HPV was compared to PreservCyt® solution (Cytyc). Evaluation was performed by testing 441 patient cervical samples collected in Corporation, Marlborough, MA). High-risk HPV status of cervical samples is obtained commercially from the Abbott RealTime HR HPV assay (Abbott Laboratories, Abbott Park, IL) and the hc2 high-risk HPV DNA test ("HC2") (Qiagen, Inc., Valencia, CA). Possible) and by further analysis of samples of inconsistent results using the LINEAR ARRAY HPV Genotyping Test (commercially available from Roche Diagnostics, Basel, CH; “Linear Array”). All three of these assays or tests were performed according to the manufacturer's instructions. A total of 227 samples were detected by both assays and 179 samples were not detected by either assay. The results of 35 inconsistent samples were analyzed by Linear Array. Of the 238 positive samples, 231 were detected by the Abbott RealTime HR HPV assay and 234 were detected by HC2. Of the 203 analyzed negative samples, 203 were not detected by the Abbott RealTime HR HPV assay and 179 were not detected by HC2. As shown in Table 8 below, the sensitivity of the Abbott RealTime HR HPV assay for the detection of HR HPV was 97% and that of the HC2 assay was 98%. The specificity of the Abbott RealTime HR HPV assay was 100% and the specificity of the HC2 assay was 88%.

  Those skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects of the invention and achieve the ends and advantages described, as well as those inherent therein. The molecular complexes and methods, procedures, treatments, molecules, specific compounds described herein are presently representative of the preferred embodiments, are exemplary, and are within the scope of the present invention. It is not intended to limit. It will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention.

  All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are hereby incorporated by reference as if each individual publication was specifically and individually indicated to be incorporated herein by reference.

  The invention described herein by way of example can be suitably practiced without any elements or limitations not specifically disclosed herein. The terms and phrases used are intended to be illustrative rather than restrictive, and in the use of such terms and expressions, any of the features shown and described or their equivalents in part Is not intended to be excluded. It will be appreciated that various modifications are possible within the scope of the claimed invention. Accordingly, it is to be understood that the present invention has been specifically disclosed by the preferred embodiments. Optional features, modifications and variations of the concepts disclosed herein may be left to those skilled in the art, and such modifications and variations are defined by the invention as defined by the appended claims. Is considered within the scope of

Claims (10)

A primer set for amplifying HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 in a test sample,
(A) three forward primers having the sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 or their complements; and (b) two having the sequences of SEQ ID NO: 4 and SEQ ID NO: 5 or their complements. Primer set including reverse primer. A primer and probe set for detecting HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 in a test sample,
(A) Three forward primers having the sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 or their complements, and two reverse primers having the sequences of SEQ ID NO: 4 and SEQ ID NO: 5 or their complements And (b) SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, sequence A primer and probe set comprising 14 probes having the sequence of SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, or their complements. A method for detecting one or more of HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 in a test sample comprising:
(A) Three forward primers having the sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 or their complements, and two reverse primers having the sequences of SEQ ID NO: 4 and SEQ ID NO: 5 or their complements And a test sample are contacted under amplification conditions to generate a first target sequence; and (b) the first target sequence and HPV 16, 18, 31, 33, 35, 39, 45 in the test sample. , 51, 52, 56, 58, 59, 66 and 68, detecting hybridization between 14 probes as an indicator of the presence of one or more, wherein the 14 probes are sequenced SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, arrangement No. 18, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21 or step having the sequences of their complements,
Including a method.   4. The method of claim 3, wherein each probe is labeled with a detectable label.   5. The method of claim 4, wherein the detectable label comprises a fluorescent moiety attached to the 5 'end of each probe.   6. The method of claim 5, wherein each probe further comprises a quenching moiety attached to its 3 'end. Furthermore,
(A) contacting a forward primer having the sequence of SEQ ID NO: 6 or its complement and a reverse primer having the sequence of SEQ ID NO: 7 or its complement with a test sample under amplification conditions for generating a second target sequence And (b) detecting hybridization between the second target sequence and a probe having the sequence of SEQ ID NO: 22 or its complement as an indicator of the presence of human beta globin in the test sample, The method of claim 3. A kit for detecting HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 in a test sample,
(A) three forward primers having the sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 or their complements;
(B) two reverse primers having the sequences of SEQ ID NO: 4 and SEQ ID NO: 5 or their complements,
(C) amplification reagents , and
(D) SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 , SEQ ID NO: 20 and SEQ ID NO: 21, or a probe having the sequence of their complements . A primer and probe kit for detecting HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 in a test sample, comprising:
(A) Three forward primers having the sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 or their complements, and two reverse primers having the sequences of SEQ ID NO: 4 and SEQ ID NO: 5 or their complements ,
(B) SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 A primer and probe kit comprising: 14 probes having the sequences of SEQ ID NO: 20 and SEQ ID NO: 21, or their complements; and (c) an amplification reagent. A kit further comprising a forward primer having a sequence of SEQ ID NO: 6 or its complement and a reverse primer having a sequence of SEQ ID NO: 7 or its complement, for detecting human beta globin in a test sample; The primer and probe kit according to claim 9 , comprising a probe having a sequence of its complement.

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